Fatty acid uptake in Candida tropicalis: induction of a ...

Fatty acid uptake in Candida tropicalis: induction of a saturable process BERNARDOL. TRIGATTI, ANDREWD. BAKER,KRISHANRAJARATNAM,RICHARDA. RACHUBINSKI, AND

GERHARD E.

GERBER'

Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by China University of Petroleum on 06/05/13 For personal use only.

Department of Biochemistry, McMaster University, Hamilton, Ont., Canada L8N 325 Received July 5, 1991 TRIGATTI,B. L., BAKER,A. D., RAJARATNAM, K., RACHUBINSKI, R. A., and GERBER,G. E. 1992. Fatty acid uptake in Candida tropicalis: induction of a saturable process. Biochem. Cell Biol. 70: 76-80. The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and V , , values of 56 p M and 15 nmol/(min.mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate. Key words: acyl-CoA synthetase, Candida tropicalis, induction, oleate, uptake.

K., RACHUBINSKI, R. A., et GERBER,G. E. 1992. Fatty acid uptake TRIGATTI, B. L., BAKER,A. D., RAJARATNAM, in Candida tropicalis: induction of a saturable process. Biochem. Cell Biol. 70 : 76-80. Les taux d'incorporation de I'oltate par les cellules de Candida tropicalis croissant en presence d'une concentration elevte d'oltate (oltate 3,s mM en prtsence de Brij 58 0,50%) sont plus Clevis que ceux obsemts dans les cellules cultivtes sur le glucose; cependant, l'incorporation de I'oltate n'est pas saturable avec la concentration du substrat. Les cellules cultivtes en prtsence d'une faible concentration d'oltate (oltate 1,O mM et Brij 58 2,5%) croissent avec une densitt plus faible et a une vitesse ltgkrement plus lente; ces cellules absorbent I'oltate a un taux 43 fois plus Clevt que les cellules en prtsence d'une forte concentration d'oltate. De plus, l'absorption de I'oltate par les cellules cultivtes dans un milieu contenant peu d'oltate est un processus saturable avec une valeur de Kt de 56 ,uM et une V,, de 15 nmol/(min mg de prottine cellulaire). La croissance de C. tropicalis en prtsence d'une faible concentration d'acides gras entraine donc nettement l'induction d'un processus d'absorption saturable. Le taux global de l'activitt de I'acylCoA synthttase dans les cellules croissant en prtsence de faibles concentrations d'oltate est deux fois plus tlevt que dans les cellules en prtsence de beaucoup d'oltate ou de glucose. Le taux de cette enzyme n'est donc pas responsable du processus saturable et suggtre que l'enzyme pourrait Ctre contrSlte in vivo ou qu'une enzyme encore non identifite serait induite par la croissance en prtsence de faibles concentrations d'oltate. Mots clks : acyl-CoA synthttase, Candida tropicalis, induction, oltate, absorption. [Traduit par la rtdaction]

The mechanism of cellular uptake of long-chain fatty acids is poorly understood. A major controversy is whether fatty acid permeation across the plasma membrane is protein mediated or simply a diffusive flip-flop process. In Escherichia coli, two gene products have been shown to be required for the process: the product of thefadL gene, which is required for the movement of fatty acids across the outer membrane which is normally impermeable to hydrophobic compounds (Black et al. 1987), and the fadD gene product, a long-chain acyl-CoA synthetase which recently has been implicated in facilitating the movement of long-chain fatty acids across the inner membrane (Mangroo and Gerber 1991). In mammalian cells, several lines of evidence support the proposal that the process is protein mediated. In hepatocytes, kinetic evidence indicates that uptake is saturable with substrate concentration (Stremmel et a/. 1986). A 40-kDa protein was identified and found to bind fatty acids with high affinity, and antibodies to this protein were reported to partially inhibit oleate uptake (Stremmel et a/. 1985, 1986). Recent evidence, however, indicates that this protein may be located in mitochondria, bringing its ABBREVIATIONS: Brij 58, polyoxyethylene 20 cetyl ether; ODm, optical density at 660 nm. ' ~ u t h o rto whom all correspondence should be addressed. t i n t e d in Canada / Imprime au Canada

involvement in fatty acid movement across the plasma membrane into question (Berk et al. 1990). Furthermore, studies using model membrane systems have suggested that fatty acid movement across the plasma membrane may be rapid (Broring et al. 1989) and not protein mediated (Doody et al. 1980; Storch and Kleinfeld 1986). Thus while it is clear that fatty acid uptake generally occurs via a saturable process, it remains to be established whether this is due to proteins involved in translocation or some other metabolic process. The yeast Candida tropicalis grows on both n-alkanes and long-chain fatty acids, with a resultant induction of peroxisomes containing the metabolic machinery for P-oxidation (Kawamoto et al. 1978; Dornrnes et al. 1983). A concomitant induction in long-chain fatty acid uptake would greatly facilitate the identification of components involved in the process; however, no studies of such inducible processes in oleate uptake have been reported. It was the purpose of this study to determine whether such inducible mechanisms for oleate uptake existed in C . tropicalis. It was found that the level of oleate uptake was dependent on the nature and concentration of the carbon source. Cells grown on high concentrations of oleate (3.5 mM oleate and 0.50% Brij 58) had low levels of nonsaturable oleate uptake, while growth of cells on a low oleate

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concentration (1.0 m M oleate and 2.5% Brij 58) resulted

in the induction of a saturable oleate uptake process. These results suggest that a protein-mediated process required for the uptake of low concentrations of fatty acid was induced by growth o n a low concentration of oleate.


Materials and methods Materials Nonradioactive oleic acid was purchased from J.T. Baker Co., while [9,10-~~]oleic acid was purchased from New England Nuclear. Yeast lytic enzyme (100 000 U/g) was obtained from ICN Biochemicals Inc. Membrane filters (type HV; pore size, 0.45 pm) were purchased from Millipore. Aqueous counting scintillant was from Amersham. All other reagents were purchased from Sigma Chemical Co. Yeast strain and culture conditions Candida tropicalis (Castellani) Berklout strain pK233 (ATCC 20336) (Tanabe et al. 1966) grown for 24 h at 30°C on malt-agar slants (3.0% malt extract and 1.5% agar) was used to inoculate 100 mL of growth medium, which consisted of phosphate buffer (21 mM (NHJ2HP04, 12 mM KH2P04, and 28 mM K2HP04, pH 7.4) containing 4.1 mM MgS0,.7H20, 74 pM FeC13.6H,0, 0.10% corn steep liquor, Brij 58, and a carbon source at the specified concentrations (Kawamoto et al. 1978). The carbon sources used were 0.10 M glucose or the potassium salt of oleate (either 1.0 mM oleate containing 2.5% Brij 58 or 3.5 mM oleate and 0.50% Brij 58, referred to as low and high oleate medium, respectively). Cultures were grown aerobically at 30°C in 250-mL Erlenmeyer flasks in an orbital shaker (170 revolutions/ min). Cells in the midexponential phase of growth were subcultured to an appropriate density in fresh medium. Cell densities were determined by measuring the optical densities at 660 nm (ODm) of 10-fold dilutions using a Cary 210 spectrophotometer and were expressed as the OD, of the undiluted culture. Oleate uptake assay Cells were harvested during their midexponential phase of growth by centrifugation at 500 x g for 10 rnin at 4.0°C, using a SS34 rotor. They were then washed twice with chilled phosphate buffer containing 0.50% Brij 58 (detergent wash buffer) and twice with chilled phosphate buffer. Centrifugation was as described above. The pellets were resuspended in chilled phosphate buffer to 1.0 x 10' cells/mL and kept on ice until used. Cells were incubated at 30°C for 10 min and the assay was begun by the addition of an equal volume of a solution containing [9,10-~~]oleate (potassium salt) and 1.0% Brij 58, which was preequilibrated to 30°C. The typical assay contained 5.0 x 10' cells/mL, 50 pM 1.0 mM [9,10-3~]oleate,and 0.50% Brij 58 (to solubilize the fatty acid) in phosphate buffer. At various times (1, 3, and 5 min), 200-pL aliquots of the assay mixture were added to 5.0 mL of icecold detergent wash buffer and filtered rapidly. The filters were washed three times with 5.0 mL of ice-cold detergent wash buffer and soaked overnight in 10 mL of scintillant prior to scintillation counting using a Beckman LS 7800 scintillation counter. Filter blanks (lacking cells) were routinely included. It was found that the washing conditions described reduced the amount of radioactive oleate nonspecifically adsorbed to the filters to levels that were insignificant relative to the uptake signal. The rates of oleate uptake at various oleate concentrations were normalized to total cellular protein content (Lowry et al. 1951) and modeled where applicable, according to the Michaelis-Menten equation by least-squares regression analysis using the EZ-Fit curve fitting program developed by Perrella (1988). Acyl-CoA synthetase assay Cells were harvested from 100 mL of growth medium and washed as described above, with the exception of the final wash which was done with ice-cold digestion buffer (50 mM KCI,

TIME (hours) FIG. 1. Growth of C. tropicalis on medium containing high or low oleate concentrations. Cells were grown in 100 mL of high oleate medium (3.5 mM oleate and 0.50% Brij 58) (a) or low oleate medium (1.0 mM oleate and 2.5% Brig 58) ( 0 )as described in Materials and methods. Aliquots of cells were removed at various time intervals and diluted 10-fold with fresh medium and their OD, was measured with a Cary 210 spectrophotometer. 5.0 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2). The pellets were resuspended to 10 mL with digestion buffer containing 2.0 mM dithiothreitol and 0.10 m M phenylmethylsulfonylfluoride, and then converted to spheroplasts by incubation at 30°C for 60 rnin with 1.0 mg yeast lytic enzyme/g wet weight cells. Spheroplasts were collected by centrifugation for 10 rnin at 4.0°C at 3000 x g with an SS34 rotor and resuspended to 5.0 mL in chilled 10 mM Tris-HC1 (pH 7.4). They were then homogenized with a tight-fitting glass-teflon homogenizer using 10 up-and-down strokes at moderate speed. Standard conditions for the acyl-CoA synthetase assay consisted of 0.10 M Tris-HC1 (pH 7.9, 10 mM MgCI,, 5.0 mM dithiothreitol, 15 mM ATP, 1.0 mM CoA, 0.20 mM [9,10-~~]0leate (potassium salt), and 0.10% Triton X-100 (Mishina et al. 1978). This was incubated for 5 min at 25"C, after which an aliquot of the crude homogenate was added to bring the total volume up to 500 pL and the protein concentrations to between 0.043 and 0.16 mg/mL. At various times 100-pL portions of the assay mixture were quenched in 500 pL of Dole solution (isopropanol - n-heptane - 1 N H2S04, 40: 10: 1 by volume). Oleoyl-CoA was recovered as described by Leblanc and Gerber (1984). Briefly, after the addition of 300 pL each of n-heptane and water to bring about phase separation, the upper organic phase was discarded, the lower phase was washed four times with 1.0 mL of diethyl ether and added to 10 mL of scintillant, and the radioactivity was determined by scintillation counting. Results were normalized to total cellular protein content (Lowry et al. 1951).

Results Growth of C. tropicalis o n oleate was highly dependent

on the concentrations of oleate a n d Brij 58 in the medium (Fig. 1). In the presence of 3.5 m M oleate a n d 0.50% Brij 58 (high oleate), the cells grew with a doubling time of 1.9 h


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[OLEATE] (mM)

FIG.3. Effect of substrate concentration on oleate uptake by C. tropicalis grown on oleate. [9,10-'~10leate uptake rates were measured at various oleate concentrations in the presence of 0.50% Brij 58, as described in Materials and methods for C. tropicalis cells grown on either low oleate medium ( 0 ) or high oleate medium (a). Results are expressed as the average rate k standard error (normalized to total cellular protein content), determined from three time courses of 1.0, 3.0, and 5.0 min. (Fig. 3). For example, at 100 pM oleate, the rate of uptake by cells grown on low oleate was approximately 43-fold that by cells grown on high oleate (9.9 compared with FIG. 2. Time courses of oleate uptake by C. tropicalis grown , for oleate 0.23 nmol/(min-mg cell protein)). The V on oleate or glucose. Cells harvested at the mid-log phase of growth uptake by low oleate grown cells was 15 1.5 nmol/ from low oleate medium (o),high oleate medium (a),1.0 mM (min-mg cell protein), while the Kt was 56 27 pM. glucose (o),or 1.0 mM lucose and 2.5% Brij 58 (a) were Acyl-CoA synthetase converts fatty acids to thioesters of incubated with 250 pM [ fHloleate in the presence of 0.50% CoA, thereby activating them towards further metabolism. Brij 58. Oleate uptake was measured as described in Materials and methods. Results were normalized to total cellular protein content It can therefore be considered to be the first enzyme involved and represent the average k standard error of three in fatty acid metabolism. Using a radiochemical assay (see determinations. Materials and methods), the time courses of activation of [9,10-3~]oleateby crude homogenates of cells were determined for cells grown on low oleate, high oleate, or glucose and reached a maximum OD660of between 3.7 and 4.0. In (Fig. 4). The accumulation of [ 9 , 1 0 - ~ ~ ] 0 l e o y l -pro~o~ the presence of 1.O mM oleate and 2.5% Brij 58 (low oleate), ceeded at a linear rate for up to 4.0 min in each case. The the cells grew with a slightly longer doubling time of 2.2 h rate of oleate activation by crude homogenates of cells grown and reached a maximum OD660of approximately 2.0. The effects of the carbon source on [9,10-3~]oleate on low oleate was higher than that by crude homogenates of cells grown on high oleate or glucose (5.9 compared with uptake were studied using a rapid filtration assay, as 3.1 nmol oleoyl-CoA/(min mg protein), respectively), described in Materials and methods (Fig. 2). Filter blanks, lacking cells, were routinely included and were insignificant indicating that a 1.9-fold induction of acyl-CoA synthetase activity resulted from growth on low oleate. compared with uptake values. The rate of oleate uptake by cells grown on glucose was low and was not increased by Discussion the presence of 2.5% Brij 58 during growth. The rate of oleate uptake by cells grown in high oleate medium was It is well known that growth of C. tropicalis on oleate higher and linear over 5 min, while the highest rates were as the sole carbon source results in the induction of peroxiby cells grown in low oleate medium. somes containing the enzymatic machinery necessary for This difference in the level of oleate uptake was further P-oxidation of long-chain fatty acids (Kawamoto et al. 1978; investigated by determining the uptake rates at various conDommes et al. 1983). The induction of fatty acid uptake centrations of substrate by cells grown in the high or low under similar conditions, however, has not been studied. oleate media (Fig. 3). Uptake rates by cells grown in high Escherichia coli, another organism in which fatty acid oleate medium increased linearly with increasing concentrametabolism is induced by growth on oleate, does contain tions of [9,10-3~]oleate.In contrast, rates of oleate uptake an inducible fatty acid uptake system (Nunn 1986). The by cells grown in low oleate medium were saturable with occurrence of such a process in C. tropicalis would greatly the [9,10-~~]oleate concentration in the uptake assay and facilitate the study of the mechanism(s) by which long-chain much higher than those by cells grown in high oleate medium fatty acid uptake occurs in eukaryotic cells. TIME (min)

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TIME (min) F I G . 4. Oleoyl-CoA synthetase activity in total homogenates of C. tropicalis cells. Spheroplasts were prepared from cells grown on low oleate medium (o), high oleate medium (o), or 1.0 mM glucose ( 0 ) .Acyl-CoA synthetase activities in total homogenates were measured as described in Materials and methods and normalized to total cellular protein content. Results are expressed as the average k standard error of triplicate samples.

It was found that a decrease in the concentration of oleate (from 3.5 to 1.0 mM) and an increase in the concentration of Brij 58 (from 0.50 to 2.5%) during growth resulted in a large induction of a saturable oleate uptake (Fig. 2). The Kt for this was found to be 56 & 27 pM. The reason for the high variability in this value is unclear; it may reflect the multistep nature of the uptake process. It is, however, clearly distinct from the nonsaturable process observed in cells grown in high oleate medium. In the low oleate medium the Brij 58 concentration was increased to further decrease the concentration of oleate monomer, owing to the formation of mixed micelles between oleate and Brij 58; the increased concentration of Brij 58 that has been shown to result in the induction of peroxisomes (Small et al. 1987) was shown not to induce oleate uptake directly, since the presence or absence of Brij 58 during growth on glucose had no effect on the rate of oleate uptake (Fig. 2). The difference in oleate uptake between cells grown on low or high oleate medium was, furthermore, not due to simple physical effects of the fatty acid on the cell membrane, because uptake assay conditions were identical for both cases. The rate-limiting step in oleate uptake appeared to change from a nonproteinmediated process, when cells were grown in the presence of a high concentration of oleate monomer, to a saturable, protein-mediated process, when cells were grown in the presence of a low concentration of oleate monomer. An earlier study (Kohlwein and Paltauf 1983) of fatty acid uptake in the related yeasts Saccharomyces uvarum and Saccharomycopsis lipolytica used cells grown on glucose; under

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these conditions inducible mechanisms for oleate uptake would have been inoperative. The present study is thus the first demonstration that the level of oleate uptake, as well as the nature of the process, is modulated by the oleate concentration during growth. The inducible nature of oleate uptake in C. tropicalis should facilitate the identification of components involved in the process. Induction of peroxisomes containing fatty acid P-oxidation enzymes was not responsible for the induction of saturable oleate uptake for the following reasons. Growth of C. tropicalis on oleate or on glucose in the presence of Brij 58 results in the induction of peroxisomes containing enzymes involved in fatty acid P-oxidation (Kawamoto et al. 1978; Dommes et al. 1983; Small et al. 1987). The induction of saturable oleate uptake was observed only in cells grown on low oleate medium, not in cells grown on high oleate medium or on glucose in the presence of Brij 58. Another possibility is that acyl-CoA synthetase is involved in the saturable uptake process. This enzyme catalyzes the first step in fatty acid metabolism: the activation of the fatty acid to its CoA thioester (Nunn 1986). Recent evidence suggests that fatty acid movement across the plasma membrane of E. coli is driven by this enzyme (Mangroo and Gerber 1991). Growth of C. tropicalis on low oleate compared with high oleate or glucose resulted in a 1.9-fold increase in the total acyl-CoA synthetase activity in cell homogenates (normalized to total cellular protein). This clearly does not account for the 43-fold difference observed in oleate uptake (also normalized for total cellular protein), suggesting either that the acyl-CoA synthetase activity is modulated in vivo or that some other as yet unidentified enzyme is induced and is responsible for the increased oleate uptake observed. We have demonstrated, therefore, that C. tropicalis is an ideal organism in which to study long-chain fatty acid uptake, since it can be made to switch from a nonsaturable to a saturable (protein mediated) oleate uptake process by manipulation of the oleate concentration during growth. This should allow for the design of experiments aimed at determining the nature of this event, which will further the understanding of the process of long-chain fatty acid uptake by eukaryotic cells.

Acknowledgements This work was supported by a Medical Research Council of Canada grant MA-6488 to G.E.G. and a Natural Sciences and Engineering Research Council of Canada grant A-3052 to R.A.R.. B.L.T. was the recipient of a Medical Research Council of Canada studentship. Berk, P.D., Wada, H., Horio, Y., et al. 1990. Plasma membrane fatty acid-binding protein and mitochondrial glutamicoxaloacetic transaminase of rat liver are related. Proc. Natl. Acad. Sci. U.S.A. 87: 3484-3488. Black, P.N., Said, B., Ghosn, C.R., et al. 1987. Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. J. Biol. Chem. 262: 1412-1419. Broring, K., Haest, C.W.M., and Deuticke, B. 1989. Translocation of oleic acid across the erythrocyte membrane: evidence for a fast process. Biochim. Biophys. Acta, 986: 321-331. Dommes, P., Dommes, V., and Kunau, W.H. 1983. &Oxidation in Candida tropicalis. Partial purification and biological function

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Kawamoto, S., Nozaki, C., Tanaka, A., and Fukui, S. 1978. Fatty acid P-oxidation system in microbodies of n-alkane grown Candida tropicalis. Eur . J . Biochem. 83: 609-6 13. Kohlwein, S.D., and Paltauf, F. 1983. Uptake of fatty acids by the yeasts Saccharomyces cerevisiae and Saccharomycopsis lipolytica. Biochim. Biophys. Acta, 792: 310-317. Leblanc, P., and Gerber, G.E. 1984. Biosynthetic utilization of photoreactive fatty acids by rat liver microsomes. Can. J. Biochem. Cell Biol. 62: 375-378. Lowry, O.H., Rosebrough. N. J., Farr, A.L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Mangroo, D., and Gerber, G.E. 1991. Fatty acid uptake in E. coli: recruitment of acyl-CoA synthetase to the plasma membrane. FASEB J. 5: 6602. Mishina, M., Kamyrio, T., Tashiro, S., and Numa, S. 1978. Separation and characterization of two long chain acyl-CoA synthetases from Candida lipolytica. Eur. J . Biochem. 82: 347-354.

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